Increased lung vascular endothelial permeability leads to the leakage of fluid and plasma proteins into lung tissue, a primary factor in the pathogenesis of Acute Lung Injury (ALI). Our studies will investigate the fundamental question that Ca2+ entry mediated by transient receptor potential cation channel 6 (TRPC6), a receptor-operated channel (ROC), is a critical determinant of increased endothelial permeability. We will test the hypothesis that TRPC6 activation is required and sufficient for endothelial contraction that mediates increased lung vascular permeability. The proposed studies will use the agonist thrombin, which activates protease activating receptor (PAR-1) on the endothelial cell surface and produces diacylglycerol, the direct activator of the TRPC6 channel. Studies will determine the role of dual lipid/protein phosphatase PTEN in the mechanism of TRPC6 activation. We will address the role of TRPC6-mediated increase in intracellular Ca2+ as a requirement for signaling the activation of RhoA and MLCK-L, and thereby promoting endothelial cell contraction. Our Specific Aims are to determine 1) role of endothelial ROC, TRPC6 in mediating Ca2+ signaling dependent increases in lung microvascular permeability, 2) PTEN-mediated mechanism of activation of TRPC6 and its consequences in the regulation of endothelial barrier function, and 3) downstream signaling mechanisms of TRPC6-induced increase in lung endothelial permeability involving the activation of PKCa and p60Src kinase and the effectors RhoA and MLCK-L isoform. These studies will be carried out at multiple levels including utilizing endothelial cells and lungs from mice carrying deletions in specific genes. Our hope is that the proposal will provide novel insights into the development of therapeutic strategies directed against TRPC6 that will prevent lung vascular leakiness.